VIBRATIONAL SPECTROSCOPY (FTIR-ATR AND FT-RAMAN)

A Rapid and Useful Tool for Phycocolloid Analysis

Leonel Pereira

IMAR-CMA, Institute of Marine Research

Department of Botany, FCTUC, University of Coimbra, P-3004-516 Coimbra, Portugal

Ana M. Amado

Molecular Physical Chemistry Group, Department of Chemistry, FCTUC, University of Coimbra

P-3004-535 Coimbra, Portugal

Paulo J. A. Ribeiro-Claro

Department of Chemistry – CICECO, Universty of Aveiro, P-3810-193 Aveiro, Porugal

Fred van de Velde

Wageningen Centre for Food Sciences, P.O. Box 557, 6700 AN Wageningen

NIZO food research, Texture Department, Kernhemseweg 2, P.O. Box 20, 6710 BA Ede, The Netherlands

Keywords: Polysaccharides, Seaweed, Phycocolloid, Carrageenan, FTIR-ATR, FT-Raman.

Abstract: The wide industrial application of phycocolloids (e.g. alginates, agar and carrageenans) is based on their

particular properties to form gels in aqueous solution. Recently, new spectroscopic techniques have

provided more accurate identification of the natural composition of the polysaccharides produced by these

seaweeds. With the combination of two spectroscopic techniques (FTIR-ATR and FT-Raman) it is possible

to identify the principal seaweed colloids in ground seaweed samples as in extracted material. Since the

seaweed samples receive the minimum of handling and treatment (e.g. they are simply dried and ground),

the composition determined represents, as accurately as possible, the native composition of the

phycocolloids.

1 INTRODUCTION

Many seaweeds produce hydrocolloids, associated

with the cell wall and intercellular spaces. Members

of the red algae (Rhodophyta) produce galactans

(e.g. carrageenans and agars) and the brown algae

(Phaeophyceae) produce uronates (alginates).

Carrageenans represent one of the major texturising

ingredients used by the food industry; they are

natural ingredients, which have been used for

decades in food, excepients applications and are

generally regarded as safe (GRAS).

The phycocolloid carrageenin, as it was first

called, was discovered by the British pharmacist

Stanford in 1862 who extracted it from Irish moss

(Chondrus crispus). The name was later changed to

carrageenan so as to comply with the ‘-an' suffix for

the names of polysaccharides. The modern

carrageenan industry dates from the 1940s, receiving

its impetus from the dairy industry where

carrageenan was found to be the ideal stabilizer for

the suspension of cocoa in milk chocolate.

The commercial carrageenans are normally

divided into three main types: kappa, iota and

lambda-carrageenan. Generally, seaweeds do not

produce these idealized and pure carrageenans, but

more likely a range of hybrid structures. Several

other carrageenan repeating units exist: e.g. xi, theta,

131

Pereira L., M. Amado A., J. A. Ribeiro-Claro P. and van de Velde F. (2009).

VIBRATIONAL SPECTROSCOPY (FTIR-ATR AND FT-RAMAN) - A Rapid and Useful Tool for Phycocolloid Analysis.

In Proceedings of the International Conference on Biomedical Electronics and Devices, pages 131-136

DOI: 10.5220/0001121301310136

 SciTePress

beta, mu and nu. The precursors (mu and nu), when

exposed to alkali conditions, are modified into kappa

and iota, respectively, through formation of the 3,6-

anhydro-galactose bridge (Rudolph, 2000).

Infrared (IR) spectroscopy was, until recently the

most frequently used vibrational technique for the

study of the chemical composition of phycocolloids.

This technique presents two main advantages: it

requires minute amounts of sample (milligrams),

and it is non-aggressive method with reliable

accuracy (Givernaud-Mouradi, 1992; Pereira et

al.,2003). However, conventional IR spectroscopy

requires laborious procedures to obtain spectra with

a good signal/noise ratio (Chopin and Whalen,

1993). This limitation was overcome with the

development of interferometric IR techniques

(associated with the Fourier transform algorithm),

known as FTIR spectroscopy (Fourier Transform

IR). More recently, Pereira and collaborators had

used a technique of analysis on the basis of FTIR-

ATR (from Attenuated Total Reflectance)

spectroscopy, allowing for the determination of the

composition of the different phycocolloids from

dried ground seaweed, without having to prepare

tablets of KBr (Pereira, 2006; Pereira and Mesquita,

2004).

In contrast to FTIR, the application of

conventional Raman spectroscopy was limited until

recently, due to need for an incident visible laser in

dispersive spectrometers: the visible laser light often

excites electronic transitions in biochemical

samples, which can lead to either sample

degradation or strong background signal from

unwanted laser-induced fluorescence. The use of

Nd:YAG lasers operating at 1064 nm (far from the

visible region) in interferometric spectrometers has

been generalized to decrease the fluorescence level

and avoid sample degradation. The modern FT-

Raman spectrometers have been used to produce

good quality Raman spectra from seaweed samples.

(Matsuhiro, 1996; Pereira et al., 2003).

In this work, a combined FTIR-ATR and FT-

Raman spectroscopy study were used to identify the

colloid produced by one of the principal source of

carrageenans, the red algae Chondrus crispus. Since

the analysis of ground seaweed samples required

minimal treatment (the seaweeds are simply dried

and ground), the determined composition represents,

as accurately as possible, the natural colloid

composition.

2 MATERIALS AND METHODS

2.1 Algal Material and Standard

Samples of Phycocolloids

Specimens of red algae (Rhodophyceae) Chondrus

crispus are collected in the central zone of the

western coast of Portugal (wild specimens) and other

are cultivated in Canada (lambda strain). Standard

samples were obtained from Sigma (type IV, C-

3889) and CP Kelco (pure lambda-carrageenan).

The sample composition and purity were

controlled by NMR.

2.2 Preparation of Ground Seaweed

Samples for FTIR-ATR and

FT-Raman

The seaweed samples were rinsed in distilled

freshwater to eliminate salt and debris from the

thallus surface and dried to constant weight at 60 ºC.

The dried seaweeds were finely ground in order to

render the samples uniform. For FTIR analysis the

samples do not need additional treatment. The

analysis by FT-Raman requires that these are

without pigmentation. The lack of pigmentation can

be achieved by sun drying (process used by

collectors/producers of commercial seaweeds) or by

pigment elimination in the laboratory by the addition

of acetone/methanol moisture (V/V) or by the

addition of calcium hypochlorite solution (4%, 30/60

s, 4 ºC) (Pereira, 2004).

2.3 Phycocolloid Extraction

Before phycocolloid extraction, the ground dry

material was rehydrated and pre-treated in acetone

followed by ethanol to eliminate the organosoluble

fraction (Zinoun and Cosson, 1996).

For extraction of the native phycocolloid, the

seaweed samples were placed in distilled water (50

ml/g), pH 7 at 85º C for 3 h. For an alkaline-

extraction (resembling the industrial method), the

samples were placed in a solution (150 mL/g) of

NaOH (1 M) at 80-85 ºC for 3-4 h according to

Pereira and Mesquita (2004), and neutralised to pH

6-8 with HCl (0.3 M).

The solutions were hot filtered, twice, under

vacuum, through cloth and glass fibre filter. The

extract was evaporated under vacuum to one-third of

the initial volume. The carrageenan was precipitated

by adding the warm solution to twice its volume of

ethanol (96 %).

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Table 1: Carrageenan composition determined by vibrational spectroscopy (FTIR-ATR and FT-Raman) and NMR.

Species/Sample Lifecycle phase Origin

Carrageenan

Yield

Alkali

extracted

(%mol)

Native

Chondrus crispus

Female

gametophyte

Portugal

(Wild)

23.2 % 70.0κ, 28.0ι κ - ι (µ/ν)

C. crispus Tetrasporophyte

Portugal

(Wild)

36.6 % 100.0 λ λ

C. crispus Tetrasporophyte

Canada

(Cultivated)

43.6 % 100.0 λ λ

Sigma - - - 100.0 λ λ

CP-Kelco - - - 100.0 λ λ

expressed in percentage of dry weight;

composition determined by

H-NMR;

composition determined by FTIR-ATR and FT-Raman

analysis of ground seaweed samples; the carrageenans are identified according to the Greek lettering system; the letters between

parenthesis ( ) correspond to the biological precursors of the carrageenans, present in native samples.

2.4 FTIR-ATR and FT-Raman

Analysis

The FTIR spectra of sample materials (ground dried

seaweed, native and alkali-modified carrageenan)

were recorded on an IFS 55 spectrometer, using a

Golden Gate single reflection diamond ATR system,

with no need for sample preparation. All spectra are

the average of two independent measurements with

128 scans each at a resolution of 2 cm

-1

The corresponding FT-Raman spectra were

recorded on a RFS-100 Bruker FT-spectrometer

using a Nd:YAG laser with an excitation wavelength

of 1064 nm. Each spectrum was the average of two

repeated measurements, with 150 scans at a

resolution of 2 cm

-1

2.5 NMR Analysis

H-NMR spectra were taken on a Bruker AMX600

spectrometer operating at 500.13 MHz at 65 °C.

Typically 64 scans were taken with an interpulse

delay of 5 s (T

values for the resonance of the

anomeric protons of κ- and ι-carrageenan are shorter

than 1.5 s). Sample preparation for the

H-NMR

experiments involved dissolving the carrageenan

sample (5 mg mL

-1

) at 80 °C in D

O containing 1

mM TSP (3-(trimethylsilyl) propionic-2,2,3,3-d

acid sodium salt) and 20 mM Na2HPO4, followed

by sonication for three times 1 h in a sonicator bath

(Branson 2510), according Pereira et al. (2007).

Chemical shifts (δ) are referred to internal TSP

standard (δ = -0.017 ppm) relative to the IUPAC

recommended standard DSS for

H according to van

de Velde et al. (2004). Assignments of the

H-NMR

spectra were based on the chemical shift data

summarized by van de Velde et al. (2002, 2004).

3 RESULTS AND DISCUSSION

The main results of the analyses are listed in Table

1. The assignments of the IR spectra were mostly

based on the previous work of Chopin et al. (1999)

and Sartori et al. (1997). The Raman spectra were

assigned based on the IR information and on the

comparison between samples of known composition,

controlled by NMR spectroscopy.

The carrageenans are identified by the Greek

lettering and by the letter code proposed by Knutsen

et al. (1994).

Figure 1 presents four different FT-Raman spectra

(Chondrus crispus, female gametophytes),

corresponding to the different tests of

depigmentation to reduce the background signal

from unwanted laser-induced fluorescence in Raman

VIBRATIONAL SPECTROSCOPY (FTIR-ATR AND FT-RAMAN) - A Rapid and Useful Tool for Phycocolloid Analysis

133

spectroscopy. The spectrum A corresponds to the

ground seaweed treated with a mixture of acetone

and methanol; this presents some fluorescence,

particularly in the spectral area 600-875 cm

-1

and the

peaks are ill-defined. The spectrum B corresponds to

the fresh seaweed treated with calcium hypochlorite

4% (30 s), then dried and milled. The spectrum C

concerns to the ground seaweed (obtained from a

herbarium sample) treated with calcium hypochlorite

4% (30 s). Finally, the spectrum D was obtained

from the native carrageenan (C. crispus water-

extracted) analysis. The last three spectra (B, C, D)

don’t present fluorescence, with peaks well-defined

and without background noise.

600 700 800 900 1000 1100 1200 1300 1400

W avenumber (cm

–1

)

805

850

600 700 800 900 1000 1100 1200 1300 1400600 700 800 900 1000 1100 1200 1300 1400

W avenumber (cm

–1

)

805

850

Figure 1: FT-Raman spectrum of ground seaweed (C.

crispus female gametophyte) treated with a mixture of

acetone and methanol (A). FT-Raman spectrum of fresh

seaweed treated with calcium hypochlorite 4% (30 s), then

dried and grounded (B). FT-Raman spectrum of ground

seaweed (obtained from a herbarium sample) treated with

calcium hypochlorite 4% (30 s) (C). FT-Raman spectrum

of C. crispus extracted carrageenan (D).

Since this algae produces a hybrid kappa/iota-

carrageenan the diagnoses peaks referenced in

Figure 1 are the 805 cm

-1

(DA2S),corresponding to

iota-carrageenan and 850 cm

-1

(G4S), corresponding

to kappa-carrageenan.

The FTIR-ATR and FT-Raman spectra of

commercial lambda-carrageenan (Sigma) and

ground C. crispus tetrasporophytes are shown in

Figure 2. These samples present high sulphate

content as indicated by the broad band between 820

and 830 cm

-1

in FTIR-ATR spectra. The C. crispus

and lambda-carrageenan FT-Raman spectra show

two combined peaks between 815 and 830 cm

-1

Figure 3 shows the FT-Raman spectra of

commercial sample (CP Kelco) of pure lambda-

carrageenan (A), alkali-extracted carrageenan (B) of

C crispus (tetrasporophyte) and ground seaweed

sample (C) of C. crispus (cultivated strain). The

spectrum of alkali-extracted carrageenan is similar

to that of commercial pure lambda-carrageenan. The

high sulphate content, typical of the lambda variant,

is patent in the spectra, with a presence of two

combined peaks at 815 cm

-1

(G/D6S) and 830 cm

-1

(G/D2S).

830

820

500 600 700 800 900 1000 1100 1200 1300 1400 1500

W avenumber (cm

-1

)

830

820

500 600 700 800 900 1000 1100 1200 1300 1400 1500500 600 700 800 900 1000 1100 1200 1300 1400 1500

W avenumber (cm

-1

)

Figure 2: FT-Raman (A) and FTIR-ATR (B) spectra of

commercial lambda-carrageenan (Sigma); FT-Raman (C)

and FTIR-ATR (D) spectra of ground seaweed sample

(Chondrus crispus, tetrasporophyte).

4 CONCLUSIONS

The present work confirms the usefulness of FTIR

spectroscopy in the comparative study of

carrageenan types. However, it also shows that the

complementary use of IR and Raman spectroscopy

provides relevant additional information, allowing a

better interpretation of the vibrational spectra and a

more accurate identification of diverse colloids and

variants. In fact, due to the different selection rules,

bands of weak intensity or even absent in the IR

spectra may appear as sharp and intense bands in the

Raman spectra. This is particularly evident, for

instance, in the spectra of different fractions

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134

belonging to the family of lambda-carrageenan

(Pereira et al., 2003) and the biological precursors of

kappa and iota-carrageenan (mu and nu,

respectively) (Pereira and Mesquita, 2004).

600 700 800 900 1000 1100 1200 1300 1400

815

830

Wavenumber (cm

-1

)

600 700 800 900 1000 1100 1200 1300 1400

815

830

Wavenumber (cm

-1

)

Figure 3: FT-Raman spectra: (A) commercial sample of

pure lambda-carrageenan (CP Kelco); Chondrus crispus

(tetrasporophyte) alkali-extracted carrageenan; (C) ground

seaweed sample (C. crispus, cultivated strain).

With the combination of these two spectroscopic

techniques (ATR-FTIR and FT-Raman), it is now

possible the rapid and reliable identification of all

major types of carrageenan, both extracted

carrageenan and grounded material samples. The

joint application of these spectroscopic techniques

has as main advantages:

a) It is a quick and simple methodology in

phycocolloid analysis. Only need few minutes,

instead of several days needed for the extraction of

colloids;

b) Requires small quantities of algal material (a

few grams of weight fresh or milligrams of dry

weight), allowing the analysis of herbarium samples,

even of algae or portions of algae with small size;

c) Since the seaweeds are subject to a process of

minimal manipulation and treatment (they are

simply dried and ground), the determined

composition represents, as accurately as possible,

the natural composition of phycocolloid produced.

Since the vibrational spectrometers are now

standard equipment in many Laboratories, the

techniques described in this work are useful for the

implementation of strategies of sustainable seaweed

harvest, the evaluation of the natural seaweed

composition with industrial potential, the evaluation

and control of the quality of the different batches of

algal material harvested and/or cultivated. These

spectroscopic techniques are also useful to analyze

the composition of pharmaceutical and cosmetic

excepients.

ACKNOWLEDGEMENTS

The authors acknowledge financial support from the

Portuguese Foundation for Science and Technology

– IMAR-CMA (Institute of Marine Research),

Unidade de Química-Física Molecular and

Laboratório Associado CICECO.

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